Identification of agents that induce E-selectin on human endothelial cells Measured in Cell-Based System Using Imaging - 2152-01_Activator_SinglePoint_HTS_Activity

Squamous cell carcinomas of the skin (SCC) are a leading cause of death in organ transplant recipients and treatment of non-melanoma skin cancers, of which SCC is the second most frequent type, account for 4.5% of all Medicare cancer costs. Immune evasion in human SCC primarily results from aberrant T cell homing. Vessels in SCC lack expression of the critical cutaneous T cell homing E-selectin more ..

Squamous cell carcinomas of the skin (SCC) are a leading cause of death in organ transplant recipients and treatment of non-melanoma skin cancers, of which SCC is the second most frequent type, account for 4.5% of all Medicare cancer costs. Immune evasion in human SCC primarily results from aberrant T cell homing. Vessels in SCC lack expression of the critical cutaneous T cell homing E-selectin and exclude the population of antigen-experienced skin homing T cells most capable of recognizing the tumor. There is therefore a need to identify novel agents that can induce endothelial activation and restore appropriate T cell homing without broad, nonspecific activation of the immune system. This project plan describes a high-throughput screening assay to identify small molecules that induce E-selectin expression on human endothelial cells. 3,000 human umbilical epithelial vein cells (HUVEC) are added to gelatin-coated imaging plates. Compounds or TNF-alpha (positive control) are added 24 hours before cells are stained for E-selectin and nuclear dyes. Cells are imaged with a 4x microIX system and the data is analyzed using MetaX software to identify the number of E-selectin positive cells. Compounds increasing the percent of E-selectin positive cells beyond 3 times the standard deviation of the neutral controls and are not greater than 20% toxic to the cells will be considered 'actives'.

Expected Outcome: Compounds that singnificantly cause an increase in E-Selectin expression and therefore have an increased number of E-selectin positive cells (gain of signal) will be considered active compounds. Compounds causing greater than 20% decrease in cell number will be excluded from the study.

PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.EXPECTED OUTCOME: Active compounds result in increasing readout signal.NORMALIZATION:The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.Experimental wells values were scaled to this range.PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.PUBCHEM_ACTIVITY_SCORE:This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .Compounds decreasing cell viability by more than 20 per cent were eliminated. The minimum PUBCHEM_ACTIVITY_SCORE of E-selectin positive cells required for a compound to be called a hit (the activity threshold, or AT) was set at 10.PERCENTAGE OF ACTIVE REPLICATES:For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 60.PUBCHEM_ACTIVITY_OUTCOME:Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_TSamples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_TSamples passing AT only were assigned an outcome of 1 (inactive) :PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_TSamples passing PAR_T only were assigned an outcome of 1 (inactive) :PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

A measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid#